The following invention relates generally to the preparation of a high specific activity thrombin enzyme from a given unit of plasma, which is sufficiently stable that it provides rapid clotting of a fibrinogen-rich solution of clotting and adhesive proteins for more than six hours when held at room temperature or lower.
Formulation of a fibrin sealant mimics the last step of the coagulation cascade wherein the enzyme thrombin cleaves fibrinogen which is then cross-linked into a semi-rigid or flexible fibrin clot. This fibrin clot adheres to wound sites, forming a barrier to fluid leaks and generates adhesion between tissues, while providing hemostatic and healing properties to the treated site.
Presently marketed, applicant""s CryoSeal(trademark) system is a device which harvests cryoprecipitated, concentrated clotting and adhesive proteins, including fibrinogen and Factor XIII from a donor""s plasma in approximately one hour. The one hour cryoprecipitation harvesting, provided by the CryoSeal(trademark) system, compared to the 1 to 2 day cryoprecipitation process routinely practiced in Blood Banks, means that CryoSeal(trademark) harvesting of clotting and adhesive proteins can occur right in the perioperative theater with the patient close by, thereby avoiding the need to initiate the process days in advance.
These CryoSeal(trademark) harvested clotting and adhesive proteins, when combined with bovine or human thrombin, forms a biological glue useful for surgical hemostasis and tissue adhesion. Commercially available thrombin, however, is generally sourced from bovine or human plasma pools, so the patient would still be at risk of negative immune reactions or contamination by infectious blood born viruses and, possibly Crutzfeld-Jacobs Disease (CJD) or new variants of CJD (NVCJD). An advantage of the CryoSeal(trademark) cryoprecipitation invention is that the harvested clotting and adhesive proteins sourced from the patient""s own blood eliminates the risk of contamination by infectious blood-borne disease when these clotting and adhesive proteins are topically applied to the patient""s surgical wound sites.
It has long been understood, however, that the safest condition for a surgical patient would result from a two component biological sealant preparation in which the thrombin component would be harvested from the same donor in which the clotting and adhesive protein component was harvestedxe2x80x94forming a fully autologous biological sealant or glue.
The concept of utilizing thrombin and/or fibrinogen sourced from the patient in a medical procedure performed on that patient is not novel and was first described by Andrianova in 1974. Some twenty years later, Cederholm-Williams PCT Patent (WO94/00566xe2x80x94Jan. 6, 1994) describes an improved fibrin glue in which the thrombin component, which required thirteen steps, including centrifugation, and separation of intermediate precipitates and adjusting the ionic strength of the blood and pH of the plasma to prepare, would be combined with a fibrinogen component also sourced from the plasma of the same donor. However, these many preparation steps are so time consuming they become impractical for use in the perioperative theater where processing times should be less than one hour.
Three years later, in 1997, Hirsh, et al. (U.S. Pat. No. 5,643,192) follows Cederholm-Williams by teaching another method of preparing fibrin glue in which both the fibrinogen and thrombin components of a fibrin glue are sourced from the same donor""s plasma. The Hirsh patent describes a method of preparing thrombin in which most of the fibrinogen in the plasma is first precipitated and removed to prepare a supernatant and then clotting the residual fibrinogen in the supernatant which is different and simpler than the method taught by Cederholm-Williams, but does not result in a commercially useful thrombin in that (see FIG. 1 of Hirsh, et al.) the thrombin provides clotting speeds of five seconds or less for only 4 minutes, and less than 10 seconds for only 47 minutes.
These clotting speeds are unsuitable to the needs of surgeons who could not plan their entire surgeries around the limitations of the Hirsh, et al. fibrin glue.
Surgeons predominately require a fast acting clotting time ( less than 5 seconds) for hemostasis and tissue sealing or adhesion. Slow clotting biological glues ( greater than 5 seconds) will often be transported away from the wound site by oozing and bleeding before they can perform their function. A surgeon utilizing the Hirsh fibrin glue would be required to arrange his surgery so that the hemostasis and tissue sealing intended for treatment with the Hirsh fibrin glue would occur within the 4 minute window where the clotting time was less than 5 seconds, making the Hirsh invention totally impractical for most surgeries which predominantly require rapid hemostasis and tissue adhesion throughout the surgery, the time span of which could extend to six hours.
The following prior art reflects the state of the art of which applicant is aware and is included herewith to discharge applicant""s acknowledged duty to disclose relevant prior art. It is stipulated, however, that none of these references teach singly nor render obvious when considered in any conceivable combination the nexus of the instant invention as disclosed in greater detail hereinafter and as particularly claimed.
Fenton, J. W., et al., xe2x80x9cHuman Thrombinsxe2x80x9d, Chemistry and Biology of Thrombin, pp. 43-70.
Rosenberg, R. D., et al., xe2x80x9cBovine Thrombin: Constant Specific Activity Products From Single Animalsxe2x80x9d, Fed. Proc., p. 321, Abstract No. 361.
Quick, A. J., et al., xe2x80x9cProduction Of Thrombin From Precipitate Obtained By Acidification Of Diluted Plasmaxe2x80x9d, pp. 114-118.
Eagle, H., xe2x80x9cStudies On Blood Coagulationxe2x80x9d, pp. 531-545, 1934.
Mann, K. G., et al., xe2x80x9cThe Molecular Weights Of Bovine Thrombin And Its Primary Autolysis Productsxe2x80x9d, pp. 6555-6557, 1969.
Mann, K. G., et al., xe2x80x9cMultiple Active Forms Of Thrombinxe2x80x9d, pp. 5994-6001, 1971.
Martin, M., et al., xe2x80x9cThrombolysis In Patients With Chronic Arterial Occlusionsxe2x80x9d, Thrombolytic Therapy, Vol. 47, pp. 235-241, 1971.
Fenton, J. W., et al., xe2x80x9cLarge-Scale Preparation And Preliminary Characterizations Of Human Thrombinxe2x80x9d, Biochimica et Biophysica Acta. Vol. 229, pp. 26-32, 1971.
Andrianova, et al., xe2x80x9cAn Accessible Method Of Simultaneous Production Of Fibrinogen And Thrombin From Bloodxe2x80x9d, pp. 648-650, 1975. (Plus English translation).
Georgi, M., et al., xe2x80x9cOcclusion Of The Renal Artery By Intra-Arterial Injection Of Thrombin In A Case Of Inoperable Renal Tumorxe2x80x9d, Deutsche Medizinische Wochenschrift, Vol. 100(47), pp. 2428-2429, 1975. (Plus English translation).
Lundblad, R. L., et al., xe2x80x9cPreparation And Partial Characterization Of Two Forms Of Bovine Thrombinxe2x80x9d, Biochemical and Biophysical Research Communications, Vol. 66(2), pp. 482-489, 1975.
Sakuragawa, N., et al., xe2x80x9cPurification And Some Characterization Of Human Thrombinxe2x80x9d, Acta Medica et Biologica, Vol. 23(1), pp. 65-73, 1975.
Fenton, J. W., et al., xe2x80x9cHuman Thrombins: Production, Evaluation, And Properties Of xcex1-Thrombinxe2x80x9d, The Journal of Biological Chemistry, Vol. 252(11), pp. 3587-3598, 1977.
Nordenman, B., et al., xe2x80x9cPurification Of Thrombin By Affinity Chromatography On Immobilized Heparinxe2x80x9d, Thrombosis Research, Vol. 11, pp. 799-808, 1977.
Nowotny, R., et al., xe2x80x9cMechanical Properties Of Fibrinogen-Adhesive Materialxe2x80x9d, Biomaterials 1980, Vol. 3, pp. 677-682, 1982.
Kotelba-Witkowska, B., et al., xe2x80x9cCryopreservation Of Platelet Concentrates Using Glycerol-Glucosexe2x80x9d, Transfusion, Vol. 22(2), pp. 121-124, 1982.
Redl, H., et al., xe2x80x9cFibrin Sealant-Antibiotic Mixturexe2x80x94Stability And Elution Behaviorxe2x80x9d, Fibrinkleber Orthop. Traumatol. Orthop. Symp., Vol. 4, pp. 178-181, 1982. (Plus English translation).
Redl, H., et al., xe2x80x9cIn Vitro Properties Of Mixtures Of Fibrin Seal And Antibioticsxe2x80x9d, Biomaterials, Vol. 4(1), pp. 29-32, 1983.
Gestring, G., et al., xe2x80x9cAutologous Fibrinogen For Tissue-Adhesion, Hemostasis And Embolizationxe2x80x9d, Vascular Surgery, Vol. 17, pp. 294-304, 1983.
Wolf, G., xe2x80x9cThe Concentrated Autologous Tissue Gluexe2x80x9d, Archives of Oto-Rhino-Laryngology, Vol. 237, pp. 279-283, 1983.
Tsvetkov, T. S., et al., xe2x80x9cA Method For Preparation Of Dry Thrombin For Topical Applicationxe2x80x9d, Cryobiology, Vol. 21(6), pp. 661-663, 1984.
Yu, X. J., et al., xe2x80x9cAffinity Chromatography Of Thrombin On Modified Polystyrene Resinsxe2x80x9d, Journal of Chromatography, Vol. 376, pp. 429-435, 1986.
Fischer, A. M., et al., xe2x80x9cThrombin Purification By One-Step Preparative Affinity Chromatography On Modified Polystyrenesxe2x80x9d, Journal of Chromatography, Vol. 363(1), pp. 95-100, 1986.
Harpel, P. C., xe2x80x9cBlood Proteolytic Enzyme Inhibitors: Their Role In Modulating Blood Coagulation And Fibrinolytic Enzyme Pathwaysxe2x80x9d, pp. 219-234, 1987.
Fenton, J. W., xe2x80x9cRegulation Of Thrombin Generation And Functionsxe2x80x9d, Seminars in Thrombosis and Hemostasis, Vol. 14(3), pp. 234-240, 1988.
Awano, K., et al., xe2x80x9cRole Of Seratonin, Histamine, And Thromboxane A2 In Platelet-Induced Contractions Of Coronary Arteries And Aortae From Rabbitsxe2x80x9d, Journal Of Cardiovascular Pharmacology, Vol. 13(5), pp. 781-792, 1989.
Mulvihill, J., et al., xe2x80x9cThrombin Stimulated Platelet Accumulation On Protein Coated Glass Capillaries: Role Of Adhesive Platelet xcex1-Granule Proteinsxe2x80x9d, Thrombosis and Haemostasis, Vol. 62(3), pp. 989-995, 1989.
Suzuki, S., et al., xe2x80x9cA Study On The Properties Of Commercial Thrombin Preparationsxe2x80x9d, Thrombosis Research, Vol. 53(3), pp. 271-277, 1989.
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DePalma, L., et.al., xe2x80x9cThe Preparation Of Fibrinogen Concentrate For Use As Fibrin Glue By Four Different Methodsxe2x80x9d, Transfusion, Vol. 33(9), pp. 717-720, 1993.
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The other prior art listed above, not all of which are specifically discussed catalog the prior art of which the applicant is aware. These undiscussed references diverge even more starkly from the instant invention specifically distinguished below.
The instant invention addresses the long felt need for a simple, practical, fast method of preparing stable human thrombin from a donor""s blood, which will provide fast clots ( less than 5 seconds) throughout a lengthy surgery (e.g. six hours) to combine with the clotting and adhesive proteins harvested and concentrated from the same unit of blood to form a biological sealant with no patient exposure to microbial or possible CJD or NVCJD contaminations. Previous works in the field (Hirsch, et al.) exemplified a thrombin with minimal stability in that the thrombin achieved rapid clotting of fibrinogen (i.e., less than 5 seconds) during only a very narrow four to five minute time period, or required so many steps and elapsed time it would not be suitable for perioperative preparation, both totally impractical for the broad range of surgeries.
The present invention provides a stable thrombin enzyme which can cause precise, repeatable fast or slow polymerization of clotting and adhesive proteins over a duration of up to six hoursxe2x80x94throughout even a long surgery. Further, the use of clotting and adhesive proteins and thrombin all sourced from a single donor will eliminate various disease risks posed from the use of commercial fibrin glues where the fibrinogen is sourced from plasma pooled from thousands of donors and the thrombin is either sourced from a similar pool of human plasma or of bovine origin. The speed and simplicity of the production of stable thrombin by use of this invention allows it to be prepared just prior to or during operative procedures and it will provide fast clotting throughout even the longest surgeries. The thrombin produced by this invention can be diluted in saline, water and a dilute CaCl2 solution (e.g. 125 mM CaCl2) to provide precise, slower clotting times thereby allowing any preferred time from less than five seconds to longer than 2 minutes.
The procedure of the invention is preferably comprised of three steps, the first two of which should preferably occur at the same time:
1. Preparing a fraction enriched in prothrombin by use of Ethanol to substantially enhance the concentration of prothrombin and at the same time remove or denature naturally occurring ingredients within plasma, such as Fibrinogen and Antithrombin III which can bind to, block, interfere with or inhibit prothrombin or its subsequent activation to long-term functional thrombin.
2. Adding calcium ions to the enriched prothrombin solution and briefly agitating the solution to convert the pro-thrombin to stable, long term thrombin.
3. Expressing the thrombin solution through a filter to remove particulate matter which would prevent spraying the thrombin through a small orifice or expressing the thrombin through a thin tube onto a wound site.
Accordingly, it is a primary object of the present invention to provide a new and novel apparatus and method to derive fast acting, stable autologous thrombin from the donor""s plasma.
It is a further object of the present invention to provide thrombin as characterized above which has a shelf life longer than most associated surgical procedures.
It is a further object of the present invention to provide thrombin as characterized above in which the clotting time can be predictably lengthened at will through dilution with saline.
It is a further object of the present invention to provide thrombin as characterized above which has simple preparatory procedures.
It is a further object of the present invention to provide a method for producing thrombin as characterized above which has a process time in as little as thirty minutes, up to seventy-five minutes.
It is a further object of the present invention to provide thrombin which can be sprayed through small orifices or expressed through thin tubes.
Viewed from a first vantage point it is the object of the present invention to provide a novel and practical method for producing stable human thrombin from a prothrombin fraction which has been substantially enriched by ethanol fractionation to increase the prothrombin concentration and at the same time remove contaminating proteins. The addition of calcium chloride to the enriched prothrombin converts prothrombin to thrombin. From the same sole donor plasma, clotting and adhesive proteins are simultaneously obtained by other means to comprise the second component necessary for the autologous biological sealant.
Viewed from a second vantage point, it is an object of the present invention to provide a method for generating autologous thrombin from a patient, the steps including: obtaining a blood product from the patient; sequestering plasma from the product; enriching the prothrombin in a plasma fraction; converting the prothrombin to thrombin, and filtering particulate from the thrombin.
Viewed from a third vantage point, it is an object of the present invention to provide a method for producing autologous thrombin which is stable for more than fifteen minutes, the steps including: sequestering pro-thrombin from plasma and converting the pro-thrombin to thrombin.
Viewed from a fourth vantage point, it is an object of the present invention to provide an autologous thrombin which provides fast clotting in less than five seconds for more than fifteen minutes.
Viewed from a fifth vantage point, it is an object of the present invention to provide a composition for extracting thrombin from plasma consisting essentially of: Plasma; Ethanol (ETOH); CaCl2.
Viewed from a sixth vantage point, it is an object of the present invention to provide a method for preparing thrombin comprising: obtaining plasma; adding ETOH and CaCl2 to the plasma, forming a composition: agitating the composition; incubating the composition in a static or rocking mode; filtering the composition of particulate, thereby passing the thrombin through the filter.
Viewed from a seventh vantage point, it is an object of the present invention to provide a device for preparing thrombin from plasma, comprising: a reaction chamber having a solution of CaCl2 and ETOH therein; means for admitting plasma into the reaction chamber; thrombin receiving syringe coupled to the reaction chamber to receive the thrombin; and a filter located between the reaction chamber and the thrombin receiving syringe.
Viewed from an eighth vantage point, it is an object of the present invention to provide an autologous biological glue processing device, comprising, in combination: a thrombin processing means, a clotting and adhesive proteins processing means operatively coupled to the thrombin processing means, means for receiving plasma via the operative coupling for subsequent conversion of the plasma to, respectively thrombin and clotting and adhesive proteins.
The present invention provides a method and apparatus that produces thrombin which is sufficiently stable that it can provide less-than-5-second clots for up to six hours, substantially more stable than demonstrated in all prior art. Further, the clot time can be modified at will through dilution with saline.
The present invention further provides an efficient method of preparation. Improved cryoprecipitation of clotting and adhesive proteins through the CryoSeal(trademark) invention requires less than one hour. In this same time frame, the autologous human thrombin component can be manufactured with minimal materials and methods from the same source plasma. Both of the biological components of the biological glue are easily combined in a surgical setting, administered to the very same donor patient, and the resultant clotting provides hemostasis or tissue adhesion at the wound site.
The present invention additionally provides a method for sterile production of both components of the biological glue. The improved sterile manufacturing described herein provides a final product that is essentially free of contamination by non autologous microbes.
These and other objects will be made manifest when considering the following detailed specification when taken in conjunction with the appended drawing figures.